Project Summary/Abstract In the last decade, Acinetobacter baumannii has emerged as one of the most highly antibiotic-resistant bacterial pathogens in the United States (US) and throughout the world. Indeed, >70% of A. baumannii clinical isolates are now extensively drug resistant (XDR; i.e. resistant to all antibiotics except colistin or tigecycline), reflecting a >15-fold increase since 2000. Infections caused by pandrug-resistant (PDR) A. baumannii (resistant to all available antibiotics) are already being seen, and will continue to increase given the lack of new drugs in the pipeline with activity against A. baumannii. In the absence of effective antibiotics, vaccination is a promising strategy to improve mortality of A. baumannii infections. We have found that vaccination with OmpA protected mice from otherwise lethal XDR A. baumannii infection (preliminary data). Antibody titers correlated with protection, immune serum enhanced opsonophagocytic killing of A. baumannii, and passive vaccination with immune serum markedly improved the survival of infected mice. Most recently, monoclonal antibodies (MAbs) were raised against OmpA from A. baumannii. Anti-OmpA MAbs effectively treated established XDR A. baumannii infection. Likewise, MAbs targeting A. baumannii capsular polysaccharide (raised by Dr. Russo (Co-I)) were effective in the treatment of A. baumannii in a wound infection model. Thus, we seek to combine MAbs directed against OmpA and capsule as passive vaccination against XDR A. baumannii. We hypothesize that an optimal regimen of MAbs will be identified that improves outcomes in rodent models of iv, lung, and wound infection. Our specific aims are to: 1) Define MAb epitopes, surface binding, and in vitro cidal mechanism against A. baumannii. These results will support selection of non-redundant, broadly active MAbs for combination testing in Aim 2; 2) Define an optimally effective combination of MAbs in iv and lung models of infection in mice, and a rat model of wound/SC infection; 3) Define the cellular and cytokine mechanisms of protection of MAb passive vaccination by selective depletion of specific host effectors (e.g., complement, macrophages, and neutrophils) during intravenous and lung infection in mice. These mechanistic results will inform future efforts to optimize the efficacy of humanized MAbs, and define surrogate efficacy assays to test in future clinical trials. A. baumannii infections are a critical unmet need for development of novel treatments. No new antibiotics to treat these infections will likely be available in the coming decade. Absent new antibiotics, MAbs are of great potential to treat such infections. A novel multivalent MAb passive vaccination strategy will be defined against A. baumannii, and mechanisms of protection will be defined by manipulating host defense effectors and modulating epitope targets. Upon completion of the proposed studies, a mixture of MAbs will be ready for humanization (funded by private capital or business grants) to support pre-clinical toxicology studies, filing an IND, and initiation of phase I clinical trials in patients with XDR/PDR A. baumannii infections.